Identification, characterization and functional expression of auxiliary activity enzymes (LPMOs) from Aspergillus fumigatus

Abstract

The brazilian sugarcane agroindustry was one of the most developed sectors in the last 4 years. Among the main productor countries, Brazil stands on the leading featuring an area greater than 9 million hectare used by sugar-alcohol sector and responding for a year production of 632 million tons. From all reductor sugar recoverable from biomass, 55.9% is destined to ethanol production and 44.1% to the commercial sugar processing. The traditional process in first generation ethanol production involves the smashing of the culms for broth obtainment, a material rich in sucrose content which is subsequently fermented by Saccharomyces cerevisiae under anaerobic conditions. However, about 30% of the mass of sugarcane is lost in the form of straw and bagasse. The lignocellulosic residue, presents composition of 32-48% cellulose, 23-27% hemicellulose and 19-32% lignin. The efficient conversion of lignocellulosic biomass to soluble sugars is a technological challenge which limits the efficient production of this material in biofuels and chemicals products. In nature, the process is due the action of a large variety of cellulases, xylanases and associated enzymes. In this context, filamentous fungi known for their ability to produce hydrolytic enzymes capable of cleaving the lignocellulosic biomass has gained prominence in recent years, including A. fumigatus. Despite its pathogenicity, A. fumigatus has highlighted a biotechnological potential related to the deconstruction of plant residues such as bagasse, making front even the T. reesei standard producer since it is considered a major producer of cellulases, hemicellulases, amylases, lignases, lipases, proteases, phosphatases and chitinase, which together have a high degree of synergy. Results obtained by our research group that characterized the secretome and the transcriptome of A. fumigatus, when cultivated in sugarcane bagasse revealed the secretion of 64 hydrolytic enzymes characterized as Carbohydrate-Active Enzymes (Cazy enzymes) and overexpression of 145 genes encoding enzymes of that group, including different Lytic Polysaccharide Monooxygenases (LPMOs). The LPMOs are classified as enzymes with Auxiliary Activities (AA) described in different bacteria and fungi and have an important role in the deconstruction of lignocellulosic biomass, since they have oxidative mechanisms of action able to boost the activity of canonical enzymes like cellulases and hemicellulases. They are classified into four main classes, and AA9, AA10 and AA11 classes are the most interest today. Recent studies, in which commercial enzyme cocktails were supplemented with LPMOs purified, showed an increase in efficiency degradation of lignocellulosic biomass, highlighting the importance of these enzymes in hydrolysis of biomass. In this context, the main objective of this work is to characterize the expression levels profile of different LPMOs of A. fumigatus on a variety of substrates and the heterologous expression of four LPMOs identified as overexpressed in these substrates where the fungus was grown. Purification and characterization of these enzymes will enable a greater understanding of the role played by them in the hydrolysis of biomass, which can help increase the efficiency of enzyme cocktails available today. (AU)